Methods and compositions for whole transcriptome amplification

Abstract

The disclosure provides for methods, compositions, systems, devices, and kits for whole transcriptome amplification using stochastic barcodes.

What is claimed is: 
     
       1. A method for labeling a plurality of nucleic acid targets from a sample, comprising:
 hybridizing the plurality of nucleic acid targets from the sample with a plurality of labeling nucleic acids each comprising a first universal label; 
 extending the plurality of labeling nucleic acids to generate a plurality of first strand polynucleotides, wherein the plurality of nucleic acid targets are single-stranded; 
 synthesizing a plurality of second strand polynucleotides using the plurality of first strand polynucleotides as templates to generate a plurality of double-stranded polynucleotides; 
 ligating an adaptor to the plurality of double-stranded polynucleotides, wherein said adaptor comprises a second universal label; and 
 amplifying the plurality of double-stranded polynucleotides using the first universal label and the second universal label as primer sequences, thereby generating a plurality of amplicons comprising the plurality of nucleic acid targets. 
 
     
     
       2. The method of  claim 1 , wherein each of the plurality of labeling nucleic acids comprises a stochastic barcode. 
     
     
       3. The method of  claim 2 , wherein said stochastic barcode comprises a molecular label, a cellular label, a target-specific region, or any combination thereof. 
     
     
       4. The method of  claim 1 , wherein the plurality of nucleic acid targets are mRNAs. 
     
     
       5. The method of  claim 4 , wherein synthesizing the plurality of second strand polynucleotides comprises nicking the plurality of mRNAs with an RNase, thereby generating one or more mRNA primers. 
     
     
       6. The method of  claim 4 , wherein the plurality of nucleic acid targets are nucleic acids from a single cell selected from the group consisting of a bacterial cell, a fungal cell, a protozoan cell, an animal cell, and a plant cell. 
     
     
       7. The method of  claim 4 , wherein the plurality of amplicons comprises a whole transcriptome amplification (WTA) product. 
     
     
       8. The method of  claim 1 , wherein each of the plurality of labeling nucleic acids is immobilized on a solid support. 
     
     
       9. The method of  claim 8 , wherein said solid support is a bead. 
     
     
       10. The method of  claim 8 , wherein at least two of said plurality of labeling nucleic acids immobilized on a single solid support comprises different molecular labels. 
     
     
       11. The method of  claim 8 , wherein said plurality of labeling nucleic acids attached to a solid support comprises the same cellular label. 
     
     
       12. The method of  claim 1 , wherein the sample comprises a single cell. 
     
     
       13. The method of  claim 1 , wherein the first universal label and the second universal label are different. 
     
     
       14. A method for labeling a plurality of nucleic acid targets from a sample comprising:
 hybridizing the plurality of nucleic acid targets from the sample with a plurality of labeling nucleic acids each comprising a first universal label; 
 extending the plurality of labeling nucleic acids to generate a plurality of first strand polynucleotides, wherein the plurality of first stand polynucleotides and the plurality of nucleic acid targets form a plurality of double-stranded polynucleotides; 
 contacting the plurality of double-stranded polynucleotides with a first transposome comprising a first transposase and a first adaptor, wherein said first adaptor comprises a second universal label; 
 ligating the first adaptor to the double-stranded polynucleotides with the first transposase to generate a plurality of double stranded polynucleotides that are ligated with the first adaptor; and 
 amplifying the plurality of double-stranded polynucleotides that are ligated with the first adaptor using the first universal label and the second universal label as primer sequences, thereby generating a plurality of amplicons comprising the plurality of nucleic acid targets. 
 
     
     
       15. The method of  claim 14 , further comprising synthesizing a plurality of second strand polynucleotides using the plurality of first strand polynucleotides as templates. 
     
     
       16. The method of  claim 14 , wherein said first adaptor comprises a stochastic barcode. 
     
     
       17. The method of  claim 14 , wherein said second universal label is a transposome sequence. 
     
     
       18. The method of  claim 14 , wherein each of the plurality of labeling nucleic acids is immobilized on a solid support. 
     
     
       19. The method of  claim 18 , wherein said solid support is a bead. 
     
     
       20. The method of  claim 19 , comprising purifying double-stranded polynucleotides that are immobilized on beads, wherein the double-stranded polynucleotides are double-stranded polynucleotides that are ligated with the first adaptor, the second adaptor, or both. 
     
     
       21. A method for labeling a plurality of nucleic acid target sequences from a single cell, comprising:
 providing the single cell to a partition comprising a solid support immobilized with a plurality of labeling nucleic acids each comprising a first universal label, wherein the single cell is a bacterial cell, a fungal cell, a protozoan cell, an animal cell, or a plant cell; 
 lysing the single cell to release the plurality of nucleic acid target sequences, wherein the nucleic acid target sequences are mRNAs; 
 hybridizing the plurality of nucleic acid target sequences from the single cell with the plurality of labeling nucleic acids; 
 extending the plurality of labeling nucleic acids to generate a plurality of first strand polynucleotides; 
 adding an adaptor sequence to the plurality of first strand polynucleotides, wherein said adaptor sequence comprises a second universal label; and 
 amplifying the plurality of first strand polynucleotides using the first universal label and the second universal label as primer sequences, thereby generating a plurality of amplicons comprising the plurality of nucleic acid target sequences. 
 
     
     
       22. The method of  claim 21 , wherein said adaptor sequence is added by a transposome. 
     
     
       23. The method of  claim 21 , wherein said adaptor is added by a ligation step.